Assessing siliceous sinter matrices for long-term preservation of lipid biomarkers in opaline sinter deposits analogous to Mars in El Tatio (Chile).

Subaerial hydrothermal systems are of great interest for paleobiology and astrobiology as plausible candidate environments to support the origin of life on Earth that offer a unique and interrelated atmosphere-hydrosphere-lithosphere interface. They harbor extensive sinter deposits of high preservation potential that are promising targets in the search for traces of possible extraterrestrial life on Hesperian Mars. However, long-term quality preservation is paramount for recognizing biosignatures in old samples and there are still significant gaps in our understanding of the impact and extent of taphonomy processes on life fingerprints. Here, we propose a study based on lipid biomarkers -highly resistant cell-membrane components- to investigate the effects of silicification on their preservation in hydrothermal opaline sinter. We explore the lipid biomarkers profile in three sinter deposits of up to ~3000 years from El Tatio, one of the best Martian analogs on Earth. The lipid profile in local living biofilms is used as a fresh counterpart of the fossil biomarkers in the centuries-old sinter deposits to qualitatively assess the taphonomy effects of silicification on the lipid's preservation. Despite the geological alteration, the preserved lipids retained a depleted stable-carbon isotopic fingerprint characteristic of biological sources, result highly relevant for astrobiology. The data allowed us to estimate for the first time the degradation rate of lipid biomarkers in sinter deposits from El Tatio, and to assess the time preservation framework of opaline silica. Auxiliary techniques of higher taxonomic resolution (DNA sequencing and metaproteomics) helped in the reconstruction of the paleobiology. The lipids were the best-preserved biomolecules, whereas the detection of DNA and proteins dropped considerably from 5 cm depth. These findings provide new insights into taphonomy processes affecting life fingerprints in hydrothermal deposits and serves as a useful baseline for assessing the time window for recovering unambiguous signs of past life on Earth and beyond.

Comprehensive Metabolic and Taxonomic Reconstruction of an Ancient Microbial Mat From the McMurdo Ice Shelf (Antarctica) by Integrating Genetic, Metaproteomic and Lipid Biomarker Analyses.

Paleobiological reconstructions based on molecular fossils may be limited by degradation processes causing differential preservation of biomolecules, the distinct taxonomic specificity of each biomolecule type, and analytical biases. Here, we combined the analysis of DNA, proteins and lipid biomarkers using 16S and 18S rRNA gene metabarcoding, metaproteomics and lipid analysis to reconstruct the taxonomic composition and metabolisms of a desiccated microbial mat from the McMurdo Ice Shelf (MIS) (Antarctica) dated ~1,000 years BP. The different lability, taxonomic resolution and analytical bias of each biomolecule type led to a distinct microbial community profile. DNA analysis showed selective preservation of DNA remnants from the most resistant taxa (e.g., spore-formers). In contrast, the proteins profile revealed microorganisms missed by DNA sequencing, such as Cyanobacteria, and showed a microbial composition similar to fresh microbial mats in the MIS. Lipid hydrocarbons also confirmed Cyanobacteria and suggested the presence of mosses or vascular plant remnants from a period in Antarctica when the climate was warmer (e.g., Mid-Miocene or Eocene). The combined analysis of the three biomolecule types also revealed diverse metabolisms that operated in the microbial mat before desiccation: oxygenic and anoxygenic photosynthesis, nitrogen fixation, nitrification, denitrification, sulfur reduction and oxidation, and methanogenesis. Therefore, the joint analysis of DNA, proteins and lipids resulted in a powerful approach that improved taxonomic and metabolic reconstructions overcoming information gaps derived from using individual biomolecules types.

Lipid Profiles From Fresh Biofilms Along a Temperature Gradient on a Hydrothermal Stream at El Tatio (Chilean Andes), as a Proxy for the Interpretation of Past and Present Biomarkers Beyond Earth.

Hydrothermal systems and their deposits are primary targets in the search for fossil evidence of life beyond Earth. However, to learn how to decode fossil biomarker records in ancient hydrothermal deposits, we must first be able to interpret unambiguously modern biosignatures, their distribution patterns, and their association with physicochemical factors. Here, we investigated the molecular and isotopic profile of microbial biomarkers along a thermal gradient (from 29 to 72°C) in a hot spring (labeled Cacao) from El Tatio, a geyser field in the Chilean Andes with abundant opaline silica deposits resembling the nodular and digitate structures discovered on Mars. As a molecular forensic approach, we focused on the analysis of lipid compounds bearing recognized resistance to degradation and the potential to reconstruct the paleobiology of an environment on a broader temporal scale than other, more labile, biomolecules. By exploiting the lipid biomarkers' potential to diagnose biological sources and carbon fixation pathways, we reconstructed the microbial community structure and its ecology along the Cacao hydrothermal transect. The taxonomic adscription of the lipid biomarkers was qualitatively corroborated with DNA sequencing analysis. The forensic capacity of the lipid biomarkers to identify biosources in fresh biofilms was validated down to the genus level for Roseiflexus, Chloroflexus, and Fischerella. We identified lipid biomarkers and DNA of several new cyanobacterial species in El Tatio and reported the first detection of Fischerella biomarkers at a temperature as high as 72°C. This, together with ecological peculiarities and the proportion of clades being characterized as unclassified, illustrates the ecological singularity of El Tatio and strengthens its astrobiological relevance. The Cacao hydrothermal ecosystem was defined by a succession of microbial communities and metabolic traits associated with a high- (72°C) to low-(29°C) temperature gradient that resembled the inferred metabolic sequence events from the 16S rRNA gene universal phylogenetic tree from thermophilic to anoxygenic photosynthetic species and oxygenic phototrophs. The locally calibrated DNA-validated lipidic profile in the Cacao biofilms provided a modern (molecular and isotopic) end member to facilitate the recognition of past biosources and metabolisms from altered biomarkers records in ancient silica deposits at El Tatio analogous to Martian opaline silica structures.

Time-Integrative Multibiomarker Detection in Triassic-Jurassic Rocks from the Atacama Desert: Relevance to the Search for Basic Life Beyond Earth.

Detecting evidence of life on other planetary bodies requires a certain understanding of known biomarkers and their chemical nature, preservation potential, or biological specificity. In a planetary search for life, carbonates are of special interest due to their known association with life as we know it. On Earth, carbonates serve as an invaluable paleogeochemical archive of fossils of up to billions of years old. Here, we investigated biomarker profiles on three Chilean Triassic-Jurassic sedimentary records regarding our search for signs of past and present life over ∼200 Ma. A multianalytical platform that combines lipid-derived biomarkers, metaproteomics, and a life detector chip (LDChip) is considered in the detection of biomolecules with different perdurability and source-diagnosis potential. The combined identification of proteins with positive LDChip inmunodetections provides metabolic information and taxonomic affiliation of modern/subrecent biosignatures. Molecular and isotopic analysis of more perdurable hydrocarbon cores allows for the identification of general biosources and dominant autotrophic pathways over time, as well as recreation of prevailing redox conditions over ∼200 Ma. We demonstrate how extraterrestrial life detection can benefit from the use of different biomarkers to overcome diagnosis limitations due to a lack of specificity and/or alteration over time. Our findings have implications for future astrobiological missions to Mars.

Fingerprinting molecular and isotopic biosignatures on different hydrothermal scenarios of Iceland, an acidic and sulfur-rich Mars analog.

Detecting signs of potential extant/extinct life on Mars is challenging because the presence of organics on that planet is expected to be very low and most likely linked to radiation-protected refugia and/or preservative strategies (e.g., organo-mineral complexes). With scarcity of organics, accounting for biomineralization and potential relationships between biomarkers, mineralogy, and geochemistry is key in the search for extraterrestrial life. Here we explored microbial fingerprints and their associated mineralogy in Icelandic hydrothermal systems analog to Mars (i.e., high sulfur content, or amorphous silica), to identify potentially habitable locations on that planet. The mineralogical assemblage of four hydrothermal substrates (hot springs biofilms, mud pots, and steaming and inactive fumaroles) was analyzed concerning the distribution of biomarkers. Molecular and isotopic composition of lipids revealed quantitative and compositional differences apparently impacted by surface geothermal alteration and environmental factors. pH and water showed an influence (i.e., greatest biomass in circumneutral settings with highest supply and turnover of water), whereas temperature conditioned the mineralogy that supported specific microbial metabolisms related with sulfur. Raman spectra suggested the possible coexistence of abiotic and biomediated sources of minerals (i.e., sulfur or hematite). These findings may help to interpret future Raman or GC-MS signals in forthcoming Martian missions.

Exploring microcystin-degrading bacteria thriving on recycled membranes during a cyanobacterial bloom.

Microcystins (MC) are highly toxic secondary metabolites produced by cyanobacterial blooms in many freshwater ecosystems used for recreational and drinking water purposes. So far, biological processes remain to be optimized for an efficient cyanotoxin removal, and new approaches are necessary to compete with physical-chemical treatments. In previous studies we provided a new concept of membrane biofilm reactor made of recycled material, in which a single MC-degrading bacterial strain was inoculated. The present study evaluates the capacity of bacterial consortia associated with freshwater cyanobacterial blooms to form biofilms on recycled membranes and remove MC. Three different discarded reverse osmosis (RO) membranes, previously used in desalination plants after treating brackish water (BWd), seawater (SWd) and brackish water but transformed into nanofiltration (BWt-NF), were exposed to a cyanobacterial bloom in San Juan reservoir (central Spain). Results showed that the three recycled membranes developed a bacterial community with MC removal capacity. Little differences in bacterial coverage and MC removal efficiency between membranes were observed after their exposure in the reservoir. High-throughput sequencing of 16S rRNA gene analysis showed similar bacterial community composition at the phylum level but dissimilar at the order level between the three membranes. This suggests possible surface selectivity on the attached bacterial community. The mlr- candidates such as Burkholderiales and Methylophilales were highly abundant in BWt-NF and BWd, respectively, while mlr+ candidates (e.g. Sphingomonadales) were low abundant in all membranes. Analysis of mlrA and mlrB genes used as markers for MC degradation following mlr-pathway confirmed the presence of this pathway in all membranes. These results suggest the co-existence of both genotypes in membrane-attached native biofilms. Therefore, this study confirms that recycled membranes are suitable support for many MC-degrading bacteria, thus giving value to discarded membranes for eco-friendly and low-cost biological filters.

Biomarker Profiling of Microbial Mats in the Geothermal Band of Cerro Caliente, Deception Island (Antarctica): Life at the Edge of Heat and Cold.

Substrate-atmosphere interfaces in Antarctic geothermal environments are hot-cold regions that constitute thin habitable niches for microorganisms with possible counterparts in ancient Mars. Cerro Caliente hill in Deception Island (active volcano in the South Shetland Islands) is affected by ascending hydrothermal fluids that form a band of warm substrates buffered by low air temperatures. We investigated the influence of temperature on the community structure and metabolism of three microbial mats collected along the geothermal band of Cerro Caliente registering 88°C, 8°C, and 2°C at the time of collection. High-throughput sequencing of small subunit ribosomal ribonucleic acid (SSU rRNA) genes and Life Detector Chip (LDChip) microarray immunoassays revealed different bacterial, archaeal, and eukaryotic composition in the three mats. The mat at 88°C showed the less diverse microbial community and a higher proportion of thermophiles (e.g., Thermales). In contrast, microbial communities in the mats at 2°C and 8°C showed relatively higher diversity and higher proportion of psychrophiles (e.g., Flavobacteriales). Despite this overall association, similar microbial structures at the phylum level (particularly the presence of Cyanobacteria) and certain hot- and cold-tolerant microorganisms were identified in the three mats. Daily thermal oscillations recorded in the substrate over the year (4.5-76°C) may explain the coexistence of microbial fingerprints with different thermal tolerances. Stable isotope composition also revealed metabolic differences among the microbial mats. Carbon isotopic ratios suggested the Calvin-Benson-Bassham cycle as the major pathway for carbon dioxide fixation in the mats at 2°C and 8°C, and the reductive tricarboxylic acid cycle and/or the 3-hydroxypropionate bicycle for the mat at 88°C, indicating different metabolisms as a function of the prevailing temperature of each mat. The comprehensive biomarker profile on the three microbial mats from Cerro Caliente contributes to unravel the diversity, composition, and metabolism in geothermal polar sites and highlights the relevance of geothermal-cold environments to create habitable niches with interest in other planetary environments.

Seasonal dynamics of microcystin-degrading bacteria and toxic cyanobacterial blooms: Interaction and influence of abiotic factors.

Massive proliferations of cyanobacteria coexist and have different interactions with other microorganisms, including microcystin (MC)-degrading bacteria. Despite their relevance in the environment for the removal of MCs, this bacterial community has been scarcely studied. The influence of physicochemical factors and the seasonal dynamics of toxic cyanobacteria on the relative abundance and seasonal dynamics of the MC-degrading bacterial community with mlr genes (mlr+) were investigated during a two-year study at a water reservoir in central Spain. The capacity of the total bacterial community on the degradation of MCs during the whole period of study was also evaluated. The results showed that the relative abundance of mlr+ bacteria started to increase after the increase in the relative abundance of toxic cyanobacteria and MC concentrations in the water, indicating a related seasonal dynamic and an important interaction between the two communities. The correspondence of several peaks of mlr+ bacteria with decreases in the relative abundance of toxic cyanobacteria and vice versa may also suggest a possible antagonistic relationship that deserves an in-depth study. The lack of a significant relationship between the physicochemical factors and the temporal shifts of both MC producers and degraders also supports the notion that the interaction of the two communities is an important driver of their seasonal dynamics in nature. Regarding the capacity of the total bacterial community for the degradation of MCs, this capacity was only observed during the toxic cyanobacterial bloom episodes, highlighting the importance of the pre-exposure to MCs in the reservoir for triggering the MC biodegradation process.

Diversity and temporal shifts of the bacterial community associated with a toxic cyanobacterial bloom: An interplay between microcystin producers and degraders.

The biodegradation of microcystins (MCs) by bacteria constitutes an important process in freshwater ecosystems to prevent the accumulation of toxins. However, little is known about the diversity and the seasonal dynamics of the bacterial community composition (BCC) involved in the degradation of MCs in nature. To explore these BCC shifts, high-throughput sequencing was used to analyse the 16S rRNA, mcyE and mlrA genes during a year in a freshwater reservoir with a toxic cyanobacterial bloom episode. The analysis of the mcyE and mlrA genes from water samples revealed the coexistence of different MC-producing and MC-degrading genotypes, respectively. The patchy temporal distribution of the mlrA genotypes (from the families Sphingomonadaceae and Xanthomonadaceae) suggests their dissimilar response to environmental conditions and the influence of other factors besides the MCs that may control their presence and relative abundance. During the maximum toxic cyanobacterial biomass and cell lysis, other bacterial taxa that lack mlr genes increased their relative abundance. Among these bacteria, those with a recognized role in the degradation of xenobiotic and other complex organic compounds (e.g., orders Myxococcales, Ellin6067, Spirobacillales and Cytophagales) were the most representative and suggest their possible involvement in the removal of MCs in the environment.

Presence or Absence of mlr Genes and Nutrient Concentrations Co-Determine the Microcystin Biodegradation Efficiency of a Natural Bacterial Community.

The microcystin biodegradation potential of a natural bacterial community coexisting with a toxic cyanobacterial bloom was investigated in a water reservoir from central Spain. The biodegradation capacity was confirmed in all samples during the bloom and an increase of mlrA gene copies was found with increasing microcystin concentrations. Among the 24 microcystin degrading strains isolated from the bacterial community, only 28% showed presence of mlrA gene, strongly supporting the existence and abundance of alternative microcystin degradation pathways in nature. In vitro degradation assays with both mlr⁺ and mlr- bacterial genotypes (with presence and absence of the complete mlr gene cluster, respectively) were performed with four isolated strains (Sphingopyxis sp. IM-1, IM-2 and IM-3; Paucibacter toxinivorans IM-4) and two bacterial degraders from the culture collection (Sphingosinicella microcystinivorans Y2; Paucibacter toxinivorans 2C20). Differences in microcystin degradation efficiencies between genotypes were found under different total organic carbon and total nitrogen concentrations. While mlr⁺ strains significantly improved microcystin degradation rates when exposed to other carbon and nitrogen sources, mlr- strains showed lower degradation efficiencies. This suggests that the presence of alternative carbon and nitrogen sources possibly competes with microcystins and impairs putative non-mlr microcystin degradation pathways. Considering the abundance of the mlr- bacterial population and the increasing frequency of eutrophic conditions in aquatic systems, further research on the diversity of this population and the characterization and conditions affecting non-mlr degradation pathways deserves special attention.

Effects of harmful cyanobacteria on the freshwater pathogenic free-living amoeba Acanthamoeba castellanii.

Grazing is a major regulating factor in cyanobacterial population dynamics and, subsequently, considerable effort has been spent on investigating the effects of cyanotoxins on major metazoan grazers. However, protozoan grazers such as free-living amoebae can also feed efficiently on cyanobacteria, while simultaneously posing a major threat for public health as parasites of humans and potential reservoirs of opportunistic pathogens. In this study, we conducted several experiments in which the freshwater amoeba Acanthamoeba castellanii was exposed to pure microcystin-LR (MC-LR) and six cyanobacterial strains, three MC-producing strains (MC-LR, MC-RR, MC-YR, MC-WR, [Dha7] MC-RR) and three strains containing other oligopeptides such as anabaenopeptins and cyanopeptolins. Although the exposure to high concentrations of pure MC-LR yielded no effects on amoeba, all MC-producing strains inflicted high mortality rates on amoeba populations, suggesting that toxic effects must be mediated through the ingestion of toxic cells. Interestingly, an anabaenopeptin-producing strain caused the greatest inhibition of amoeba growth, indicating that toxic bioactive compounds other than MCs are of great importance for amoebae grazers. Confocal scanning microscopy revealed different alterations in amoeba cytoskeleton integrity and as such, the observed declines in amoeba densities could have indeed been caused via a cascade of cellular events primarily triggered by oligopeptides with protein-phosphatase inhibition capabilities such as MCs or anabaenopeptins. Moreover, inducible-defense mechanisms such as the egestion of toxic, MC-producing cyanobacterial cells and the increase of resting stages (encystation) in amoebae co-cultivated with all cyanobacterial strains were observed in our experiments. Consequently, cyanobacterial strains showed different susceptibilities to amoeba grazing which were possibly influenced by the potentiality of their toxic secondary metabolites. Hence, this study shows the importance of cyanobacterial toxicity against amoeba grazing and, that cyanobacteria may contain a wide range of chemical compounds capable of negatively affect free-living, herbivorous amoebae. Moreover, this is of high importance for understanding the interactions and population dynamics of such organisms in aquatic ecosystems.